ABSTRACT
Recombinant adeno-associated virus (AAV) vectors have emerged as the preferred platform for gene therapy of rare human diseases. Despite the clinical promise, host immune responses to AAV vectors and transgene remain a major barrier to the development of successful AAV-based human gene therapies. Here, we assessed the human innate immune response to AAV9, the preferred serotype for AAV-mediated gene therapy of the CNS. We showed that AAV9 induced type I interferon (IFN) and IL-6 responses in human blood from healthy donors. This innate response was replicated with AAV6, required full viral particles, but was not observed in every donor. Depleting CpG motifs from the AAV transgene or inhibiting TLR9 signaling reduced type I IFN response to AAV9 in responding donors, highlighting the importance of TLR9-mediated DNA sensing for the innate response to AAV9. Remarkably, we further demonstrated that only seropositive donors with preexisting antibodies to AAV9 capsid mounted an innate immune response to AAV9 in human whole blood and that anti-AAV9 antibodies were necessary and sufficient to promote type I IFN release and plasmacytoid dendritic (pDC) cell activation in response to AAV9. Thus, our study reveals a previously unidentified requirement for AAV preexisting antibodies for TLR9-mediated type I IFN response to AAV9 in human blood.
Subject(s)
Dependovirus , Genetic Vectors , Immunity, Humoral , Interferon Type I , Toll-Like Receptor 9 , Humans , Toll-Like Receptor 9/immunology , Toll-Like Receptor 9/genetics , Dependovirus/genetics , Dependovirus/immunology , Interferon Type I/immunology , Genetic Vectors/genetics , Immunity, Innate , Dendritic Cells/immunology , Antibodies, Viral/blood , Antibodies, Viral/immunology , Genetic Therapy , Interleukin-6/blood , Interleukin-6/immunologyABSTRACT
Toll-like receptor 9 (TLR9) is an innate immune receptor that localizes to endosomes in antigen presenting cells and recognizes single stranded unmethylated CpG sites on bacterial genomic DNA (gDNA). Previous bioinformatic studies have demonstrated that the genome of the human pathogen Chlamydia trachomatis contains TLR9 stimulatory motifs, and correlative studies have implied a link between human TLR9 (hTLR9) genotype variants and susceptibility to infection. Here, we present our evaluation of the stimulatory potential of C. trachomatis gDNA and its recognition by hTLR9- and murine TLR9 (mTLR9)-expressing cells. Utilizing reporter cell lines, we demonstrate that purified gDNA from C. trachomatis can stimulate hTLR9 signaling, albeit at lower levels than gDNA prepared from other Gram-negative bacteria. Interestingly, we found that while C. trachomatis is capable of signaling through hTLR9 and mTLR9 during live infections in HEK293 reporter cell lines, signaling only occurs at later developmental time points. Chlamydia-specific induction of hTLR9 is blocked when protein synthesis is inhibited prior to the RB-to-EB conversion, exacerbated by the inhibition of lipooligosaccharide biosynthesis, and is significantly altered during the induction of aberrance/persistence. Our observations support the hypothesis that chlamydial gDNA is released during the conversion between the pathogen's replicative and infectious forms and during treatment with antibiotics targeting peptidoglycan assembly. Given that C. trachomatis inclusions do not co-localize with TLR9-containing vacuoles in the pro-monocytic cell line U937, our findings also hint that chlamydial gDNA is capable of egress from the inclusion, and traffics to TLR9-containing vacuoles via an as yet unknown pathway.
Subject(s)
Chlamydia Infections , Chlamydia trachomatis , Signal Transduction , Toll-Like Receptor 9 , Chlamydia trachomatis/immunology , Chlamydia trachomatis/metabolism , Chlamydia trachomatis/genetics , Humans , Toll-Like Receptor 9/metabolism , Toll-Like Receptor 9/genetics , Animals , Mice , Chlamydia Infections/microbiology , Chlamydia Infections/immunology , Chlamydia Infections/metabolism , HEK293 Cells , DNA, Bacterial/genetics , Cell LineABSTRACT
BACKGROUND: Promising outcomes have been observed in multiple myeloma (MM) with the use of immunotherapies, specifically chimeric antigen receptor T (CAR-T) cell therapy. However, a portion of MM patients do not respond to CAR-T therapy, and the reasons for this lack of response remain unclear. The objective of this study was to investigate the impact of miR-34a on the immunosuppressive polarization of macrophages obtained from MM patients. METHODS: The levels of miR-34a and TLR9 (Toll-like receptor 9) were examined in macrophages obtained from both healthy individuals and patients with MM. ELISA was employed to investigate the cytokine profiles of the macrophage samples. Co-culture experiments were conducted to evaluate the immunomodulatory impact of MM-associated macrophages on CAR-T cells. RESULTS: There was an observed suppressed activation of macrophages and CD4+ T lymphocytes in the blood samples of MM patients. Overexpression of miR-34a in MM-associated macrophages dampened the TLR9 expression and impaired the inflammatory polarization. In both the co-culture system and an animal model, MM-associated macrophages suppressed the activity and tumoricidal effect of CAR-T cells in a miR-34a-dependent manner. CONCLUSION: The findings imply that targeting the macrophage miR-34a/TLR9 axis could potentially alleviate the immunosuppression associated with CAR-T therapy in MM patients.
Subject(s)
MicroRNAs , Multiple Myeloma , Signal Transduction , Toll-Like Receptor 9 , Multiple Myeloma/immunology , Multiple Myeloma/genetics , Multiple Myeloma/therapy , Multiple Myeloma/metabolism , MicroRNAs/genetics , Toll-Like Receptor 9/metabolism , Toll-Like Receptor 9/genetics , Humans , Animals , Mice , Coculture Techniques , Tumor-Associated Macrophages/immunology , Tumor-Associated Macrophages/metabolism , Macrophages/immunology , Macrophages/metabolism , Immunotherapy, Adoptive/methods , Male , Female , Macrophage Activation/immunology , Macrophage Activation/genetics , Cell Line, TumorABSTRACT
The bleomycin-induced scleroderma model is a well-established and dependable method for creating a mouse model of SSc (systemic sclerosis). In the field of skin connective tissue diseases, increasing evidence from clinical and animal experiments suggests that TLRs (Toll-like receptors) play an important role in several diseases. This study aimed to determine the role of TLR7 (Toll-like receptor 7) and TLR9 (Toll-like receptor 9) in the mechanisms of immune abnormalities and fibrosis in SSc. This study used TLR7-KO mice (TLR7-knockout mice with a balb/c background) and TLR9-KO mice (TLR9-knockout mice with a balb/c background) as well as WT mice (wild-type balb/c mice). All three kinds of mice were induced by BLM (bleomycin) in a scleroderma model as the experimental group; meanwhile, WT mice treated with PBS (phosphate-buffered saline) were used as the control group. We analyzed the fibrotic phenotype and the immunological abnormality phenotype of TLR7-deficient and TLR9-deficient mice in the SSc disease model using flow cytometry, RT-PCR (reverse transcription-polymerase chain reaction), a histological examination, and IHC (immunohistochemical staining). In a mouse model of SSc disease, the deletion of TLR7 attenuated skin and lung fibrosis, while the deletion of TLR9 exacerbated skin and lung fibrosis. The deletion of TLR7 resulted in a relative decrease in the infiltration and expression of various pro-inflammatory and fibrotic cells and cytokines in the skin. On the other hand, the deletion of TLR9 resulted in a relative increase in the infiltration and expression of various pro-inflammatory and cytokine-inhibiting cells and cytokines in the skin. Under the influence of pDCs (plasmacytoid dendritic cells), the balances of Beff/Breg (IL-6 + CD19 + B cell/IL-10 + CD19 + B cell), Th17/Treg (IL-17A + CD4 + T cell/Foxp3 + CD25 + CD4 + T cell), M1/M2 (CD86 + macrophage/CD206 + macrophage), and Th1/Th2 (TNFα + CD3 + CD4 + T cell/IL-4 + CD3 + CD4 + T cell) were biased towards the suppression of inflammation and fibrosis as a result of the TLR7 deletion. Comparatively, the balance was biased towards promoting inflammation and fibrosis due to the TLR9 deletion. In the SSc model, TLR7 promoted inflammation and fibrosis progression, while TLR9 played a protective role. These results suggest that TLR7 and TLR9 play opposite roles in triggering SSc to produce immune system abnormalities and skin fibrosis.
Subject(s)
Disease Models, Animal , Mice, Knockout , Scleroderma, Systemic , Toll-Like Receptor 7 , Toll-Like Receptor 9 , Animals , Toll-Like Receptor 7/metabolism , Toll-Like Receptor 7/genetics , Scleroderma, Systemic/metabolism , Scleroderma, Systemic/pathology , Scleroderma, Systemic/immunology , Scleroderma, Systemic/genetics , Toll-Like Receptor 9/metabolism , Toll-Like Receptor 9/genetics , Mice , Bleomycin/adverse effects , Mice, Inbred BALB C , Cytokines/metabolism , Skin/pathology , Skin/metabolism , Skin/immunology , Fibrosis , Pulmonary Fibrosis/metabolism , Pulmonary Fibrosis/pathology , Pulmonary Fibrosis/etiology , Membrane GlycoproteinsABSTRACT
Fibroblastic reticular cells (FRCs) are a subpopulation of stromal cells modulating the immune environments in health and disease. We have previously shown that activation of TLR9 signaling in FRC in fat-associated lymphoid clusters (FALC) regulate peritoneal immunity via suppressing immune cell recruitment and peritoneal resident macrophage (PRM) retention. However, FRCs are heterogeneous across tissues and organs. The functions of each FRC subset and the regulation of TLR9 in distinct FRC subsets are unknown. Here, we confirmed that specific deletion of TLR9 in FRC improved bacterial clearance and survival during peritoneal infection. Furthermore, using single-cell RNA sequencing, we found two subsets of FRCs (CD55hi and CD55lo) in the mesenteric FALC. The CD55hi FRCs were enriched in gene expression related to extracellular matrix formation. The CD55lo FRCs were enriched in gene expression related to immune response. Interestingly, we found that TLR9 is dominantly expressed in the CD55lo subset. Activation of TLR9 signaling suppressed proliferation, cytokine production, and retinoid metabolism in the CD55lo FRC, but not CD55hi FRC. Notably, we found that adoptive transfer of Tlr9 -/-CD55lo FRC from mesenteric FALC more effectively improved the survival during peritonitis compared with WT-FRC or Tlr9 -/-CD55hi FRC. Furthermore, we identified CD55hi and CD55lo subsets in human adipose tissue-derived FRC and confirmed the suppressive effect of TLR9 on the proliferation and cytokine production in the CD55lo subset. Therefore, inhibition of TLR9 in the CD55lo FRCs from adipose tissue could be a useful strategy to improve the therapeutic efficacy of FRC-based therapy for peritonitis.
Subject(s)
Fibroblasts , Peritonitis , Signal Transduction , Toll-Like Receptor 9 , Animals , Humans , Male , Mice , Disease Models, Animal , Fibroblasts/metabolism , Fibroblasts/immunology , Immunomodulation , Mice, Inbred C57BL , Mice, Knockout , Peritonitis/immunology , Peritonitis/metabolism , Toll-Like Receptor 9/metabolism , Toll-Like Receptor 9/geneticsABSTRACT
Chronic obstructive pulmonary disease (COPD) is characterised by persistent airway inflammation even after cigarette smoking cessation. Neutrophil extracellular traps (NETs) have been implicated in COPD severity and acute airway inflammation induced by short-term cigarette smoke (CS). However, whether and how NETs contribute to sustained airway inflammation in COPD remain unclear. This study aimed to elucidate the immunoregulatory mechanism of NETs in COPD, employing human neutrophils, airway epithelial cells (AECs), dendritic cells (DCs), and a long-term CS-induced COPD mouse model, alongside cyclic guanosine monophosphate-adenosine monophosphate synthase and toll-like receptor 9 knockout mice (cGAS--/-, TLR9-/-); Additionally, bronchoalveolar lavage fluid (BALF) of COPD patients was examined. Neutrophils from COPD patients released greater cigarette smoke extract (CSE)-induced NETs (CSE-NETs) due to mitochondrial respiratory chain dysfunction. These CSE-NETs, containing oxidatively-damaged DNA (NETs-DNA), promoted AECs proliferation, nuclear factor kappa B (NF-κB) activation, NF-κB-dependent cytokines and type-I interferons production, and DC maturation, which were ameliorated/reversed by silencing/inhibition of cGAS/TLR9. In the COPD mouse model, blocking NETs-DNA-sensing via cGAS-/- and TLR9-/- mice, inhibiting NETosis using mitoTEMPO, and degrading NETs-DNA with DNase-I, respectively, reduced NETs infiltrations, airway inflammation, NF-κB activation and NF-κB-dependent cytokines, but not type-I interferons due to IFN-α/ß receptor degradation. Elevated NETs components (myeloperoxidase and neutrophil elastase activity) in BALF of COPD smokers correlated with disease severity and NF-κB-dependent cytokine levels, but not type-I interferon levels. In conclusion, NETs-DNA promotes NF-κB-dependent autoimmunity via cGAS/TLR9 in long-term CS exposure-induced COPD. Therefore, targeting NETs-DNA and cGAS/TLR9 emerges as a potential strategy to alleviate persistent airway inflammation in COPD.
Subject(s)
Extracellular Traps , NF-kappa B , Neutrophils , Nucleotidyltransferases , Pulmonary Disease, Chronic Obstructive , Toll-Like Receptor 9 , Pulmonary Disease, Chronic Obstructive/genetics , Pulmonary Disease, Chronic Obstructive/immunology , Pulmonary Disease, Chronic Obstructive/pathology , Toll-Like Receptor 9/genetics , Toll-Like Receptor 9/immunology , Extracellular Traps/immunology , Extracellular Traps/genetics , Nucleotidyltransferases/genetics , Nucleotidyltransferases/immunology , Animals , Humans , NF-kappa B/genetics , NF-kappa B/immunology , NF-kappa B/metabolism , Mice , Neutrophils/immunology , Neutrophils/pathology , Mice, Knockout , Autoimmunity/genetics , Male , DNA/genetics , DNA/immunology , Female , Disease Models, Animal , Middle AgedABSTRACT
Introduction: Type 1 diabetes (T1D) is an autoimmune disease characterized by the destruction of insulin-producing ß cells. Toll-like receptor 9 (TLR9) plays a role in autoimmune diseases, and B cell-specific TLR9 deficiency delays T1D development. Gut microbiota are implicated in T1D, although the relationship is complex. However, the impact of B cell-specific deficiency of TLR9 on intestinal microbiota and the impact of altered intestinal microbiota on the development of T1D are unclear. Objectives: This study investigated how gut microbiota and the intestinal barrier contribute to T1D development in B cell-specific TLR9-deficient NOD mice. Additionally, this study explored the role of microbiota in immune regulation and T1D onset. Methods: The study assessed gut permeability, gene expression related to gut barrier integrity, and gut microbiota composition. Antibiotics depleted gut microbiota, and fecal samples were transferred to germ-free mice. The study also examined IL-10 production, Breg cell differentiation, and their impact on T1D development. Results: B cell-specific TLR9-deficient NOD mice exhibited increased gut permeability and downregulated gut barrier-related gene expression. Antibiotics restored gut permeability, suggesting microbiota influence. Altered microbiota were enriched in Lachnospiraceae, known for mucin degradation. Transferring this microbiota to germ-free mice increased gut permeability and promoted IL-10-expressing Breg cells. Rag-/- mice transplanted with fecal samples from Tlr9 fl/fl Cd19-Cre+ mice showed delayed diabetes onset, indicating microbiota's impact. Conclusion: B cell-specific TLR9 deficiency alters gut microbiota, increasing gut permeability and promoting IL-10-expressing Breg cells, which delay T1D. This study uncovers a link between TLR9, gut microbiota, and immune regulation in T1D, with implications for microbiota-targeted T1D therapies.
Subject(s)
Diabetes Mellitus, Type 1 , Gastrointestinal Microbiome , Interleukin-10 , Mice, Inbred NOD , Toll-Like Receptor 9 , Animals , Toll-Like Receptor 9/deficiency , Toll-Like Receptor 9/genetics , Toll-Like Receptor 9/metabolism , Gastrointestinal Microbiome/immunology , Interleukin-10/metabolism , Mice , Diabetes Mellitus, Type 1/immunology , Diabetes Mellitus, Type 1/microbiology , Mice, Knockout , B-Lymphocytes, Regulatory/immunology , Female , B-Lymphocytes/immunology , B-Lymphocytes/metabolismABSTRACT
BACKGROUND: Inflammatory bowel diseases (IBD), Crohn's disease (CD), and ulcerative colitis (UC) are diseases that result from the combined effects of a predisposing genetic background and several environmental factors, including smoking. Some genes can influence these diseases through genetic inheritance, and their regulation is explained by gene polymorphism. However, Toll-like receptor (TLR) genes have been identified as susceptibility genes for CD and UC. METHODS: A case-control study was performed on a Turkish population composed of 105 healthy controls and 79 CD, 77 UC patients genotyped by Allele-specific PCR and PCR-RFLP for TLR9 (T-1486C) and TLR 2 (-196 to -174del) gene. Genotype and allele frequencies of TLR9 (T-1486C) and TLR 2 (-196 to -174del) gene polymorphisms compared to allele frequencies in CD and UC patients. RESULTS: No statistically significant findings were found between the CD, UC patients, and the control group in terms of both genotype distributions and allele frequencies for TLR 9 (T-1486C; rs187084) and TLR 2 (-196 to -174del; rs111200466) gene polymorphisms in a Turkish population (P > 0.05). CONCLUSION: No association was found between the TLR2 (rs111200466) and TLR 9 (rs187084) gene polymorphisms among IBD patients and the control groups in the Turkish population.
Subject(s)
Colitis, Ulcerative , Crohn Disease , Genetic Predisposition to Disease , Genotype , Inflammatory Bowel Diseases , Toll-Like Receptor 2 , Toll-Like Receptor 9 , Humans , Toll-Like Receptor 2/genetics , Case-Control Studies , Male , Female , Toll-Like Receptor 9/genetics , Adult , Crohn Disease/genetics , Inflammatory Bowel Diseases/genetics , Colitis, Ulcerative/genetics , Turkey , Gene Frequency , Middle Aged , Polymorphism, Single Nucleotide , Prognosis , Follow-Up Studies , Young AdultABSTRACT
In specific pathological conditions, addressing liver injury may yield favorable effects on renal function through the phenomenon of liver-kidney crosstalk. Mitochondrial DNA (mtDNA) possesses the capability to trigger downstream pathways of inflammatory cytokines, ultimately leading to immune-mediated organ damage. Consequently, understanding the intricate molecular mechanisms governing mtDNA involvement in diseases characterized by liver-kidney crosstalk is of paramount significance. This study seeks to elucidate the role of mtDNA in conditions marked by liver-kidney crosstalk. In previous clinical cases, it has been observed that patients with Trichloroethylene Hypersensitivity Syndrome (TCE-HS) who experience severe liver injury often also exhibit renal injury. In this study, patients diagnosed with trichloroethylene hypersensitivity syndrome were recruited from Shenzhen Occupational Disease Control Center. And Balb/c mice were treated with trichloroethylene. The correlation between liver and kidney injuries in patients with TCE-HS was assessed using Enzyme-Linked Immunosorbent Assay (ELISA). Alterations in mtDNA levels were examined in mouse hepatocytes, red blood cells (RBCs), and renal tubular epithelial cells utilizing immunofluorescence and PCR techniques. TCE-sensitized mice exhibited a significant increase in reactive oxygen species (ROS) and the opening of the mitochondrial permeability transition pore in hepatocytes, resulting in the release of mtDNA. Furthermore, heightened levels of mtDNA and Toll-like Receptor 9 (TLR9) expression were observed in RBCs. Additional experiments demonstrated elevated expression of TLR9 and its downstream mediator MyD88 in renal tubule epithelial cells of TCE-sensitized mice. In vitro investigations confirmed that mtDNA activates the TLR9 pathway in TCMK-1 cells. Collectively, these results suggest that mtDNA released from mitochondrial damage in hepatocytes is carried by RBCs to renal tubular epithelial cells and mediates inflammatory injury in renal tubular epithelial cells through activation of the TLR9 receptor.
Subject(s)
DNA, Mitochondrial , Liver , Mice, Inbred BALB C , Reactive Oxygen Species , Toll-Like Receptor 9 , Trichloroethylene , Animals , Trichloroethylene/toxicity , DNA, Mitochondrial/genetics , Humans , Liver/pathology , Liver/drug effects , Liver/metabolism , Liver/immunology , Female , Mice , Adult , Male , Reactive Oxygen Species/metabolism , Toll-Like Receptor 9/metabolism , Toll-Like Receptor 9/genetics , Hepatocytes/drug effects , Hepatocytes/metabolism , Kidney/pathology , Kidney/drug effects , Kidney/metabolism , Myeloid Differentiation Factor 88/metabolism , Myeloid Differentiation Factor 88/genetics , Middle Aged , Chemical and Drug Induced Liver Injury/immunology , Chemical and Drug Induced Liver Injury/metabolism , Drug Hypersensitivity Syndrome/immunology , Erythrocytes/drug effects , Erythrocytes/metabolism , Erythrocytes/immunologyABSTRACT
Toll-like receptor 9 (TLR9) recognizes bacterial, viral and self DNA and play an important role in immunity and inflammation. However, the role of TLR9 in obesity is less well-studied. Here, we generate B-cell-specific Tlr9-deficient (Tlr9fl/fl/Cd19Cre+/-, KO) B6 mice and model obesity using a high-fat diet. Compared with control mice, B-cell-specific-Tlr9-deficient mice exhibited increased fat tissue inflammation, weight gain, and impaired glucose and insulin tolerance. Furthermore, the frequencies of IL-10-producing-B cells and marginal zone B cells were reduced, and those of follicular and germinal center B cells were increased. This was associated with increased frequencies of IFNγ-producing-T cells and increased follicular helper cells. In addition, gut microbiota from the KO mice induced a pro-inflammatory state leading to immunological and metabolic dysregulation when transferred to germ-free mice. Using 16 S rRNA gene sequencing, we identify altered gut microbial communities including reduced Lachnospiraceae, which may play a role in altered metabolism in KO mice. We identify an important network involving Tlr9, Irf4 and Il-10 interconnecting metabolic homeostasis, with the function of B and T cells, and gut microbiota in obesity.
Subject(s)
B-Lymphocytes , Diet, High-Fat , Dysbiosis , Gastrointestinal Microbiome , Inflammation , Interleukin-10 , Mice, Knockout , Obesity , Toll-Like Receptor 9 , Animals , Obesity/immunology , Obesity/microbiology , Obesity/metabolism , Dysbiosis/immunology , Dysbiosis/microbiology , Toll-Like Receptor 9/metabolism , Toll-Like Receptor 9/genetics , B-Lymphocytes/immunology , B-Lymphocytes/metabolism , Inflammation/metabolism , Mice , Diet, High-Fat/adverse effects , Interleukin-10/metabolism , Male , Mice, Inbred C57BL , Disease Models, Animal , Interferon Regulatory FactorsABSTRACT
Myeloid-derived suppressor cells (MDSCs) are a group of heterologous populations of immature bone marrow cells consisting of progenitor cells of macrophages, dendritic cells and granulocytes. Recent studies have revealed that the accumulation of MDSCs in the mouse spleen plays a pivotal role in suppressing the immune response following JEV infection. However, the mechanisms by which JEV induces MDSCs are poorly understood. Here, it was found that JEV infection induces mitochondrial damage and the release of mitochondrial DNA (mtDNA), which further leads to the activation of TLR9. TLR9 deficiency decreases the M-MDSCs population and their suppressive function both in vitro and in vivo. Moreover, the increase of MHCâ ¡ expression on antigen-presenting cells and CD28 expression on T cells in TLR9-/- mice was positively correlated with M-MDSCs reduction. Accordingly, the survival rate of TLR9-/- mice dramatically increased after JEV infection. These findings reveal the connections of mitochondrial damage and TLR9 activation to the induction of M-MDSCs during JEV infection.
Subject(s)
Mice, Knockout , Myeloid-Derived Suppressor Cells , Toll-Like Receptor 9 , Toll-Like Receptor 9/metabolism , Toll-Like Receptor 9/genetics , Animals , Myeloid-Derived Suppressor Cells/immunology , Myeloid-Derived Suppressor Cells/metabolism , Mice , Mice, Inbred C57BL , Mitochondria/metabolism , DNA, Mitochondrial/genetics , DNA, Mitochondrial/metabolism , Monocytes/immunology , Monocytes/metabolismABSTRACT
Aging probably is the most complexed process in biology. It is manifested by a variety of hallmarks. These hallmarks weave a network of aging; however, each hallmark is not uniformly strong for the network. It is the weakest link determining the strengthening of the network of aging, or the maximum lifespan of an organism. Therefore, only improvement of the weakest link has the chance to increase the maximum lifespan but not others. We hypothesize that mitochondrial dysfunction is the weakest link of the network of aging. It may origin from the innate intramitochondrial immunity related to the activities of pathogen DNA recognition receptors. These receptors recognize mtDNA as the PAMP or DAMP to initiate the immune or inflammatory reactions. Evidence has shown that several of these receptors including TLR9, cGAS and IFI16 can be translocated into mitochondria. The potentially intramitochondrial presented pathogen DNA recognition receptors have the capacity to attack the exposed second structures of the mtDNA during its transcriptional or especially the replicational processes, leading to the mtDNA mutation, deletion, heteroplasmy colonization, mitochondrial dysfunction, and alterations of other hallmarks, as well as aging. Pre-consumption of the intramitochondrial presented pathogen DNA recognition receptors by medical interventions including development of mitochondrial targeted small molecule which can neutralize these receptors may retard or even reverse the aging to significantly improve the maximum lifespan of the organisms.
Subject(s)
Aging , DNA, Mitochondrial , Immunity, Innate , Mitochondria , Humans , Mitochondria/metabolism , DNA, Mitochondrial/genetics , DNA, Mitochondrial/metabolism , Toll-Like Receptor 9/metabolism , Toll-Like Receptor 9/genetics , Animals , Mitochondrial Proteins/metabolism , Mitochondrial Proteins/geneticsABSTRACT
IgA plays a vital role in defending against the infectious pathogens. However, the specific regulatory pathways involved in IgA secretion in the context of PEDV infection have remained elusive. Therefore, in this study, we explore the molecular mechanisms underlying IgA secretion in response to infection, with a particular focus on PEDV, a devastating enteric virus affecting global swine production. Our investigation begins by examining changes in IgA concentrations in both serum and small intestinal contents following PEDV infection in 2- and 4-week-old pigs. Remarkably, a significant increase in IgA levels in these older pigs post-infection were observed. To delve deeper into the regulatory mechanisms governing IgA secretion in response to PEDV infection, isolated porcine intestinal B cells were co-cultured with monocytes derived DCs (Mo-DCs) in vitro. In the intestinal DC-B cell co-cultures, IgA secretion was found to increase significantly after PEDV infection, as well as upregulating the expression of AID, GLTα and PSTα reflecting isotype switching to IgA. In addition, the expression of TLR9 was upregulated in these cultures, as determined by RT-qPCR and western blotting. Moreover, our findings extend to in vivo observations, where we detected higher levels of TLR9 expression in the ileum of pig post PEDV infection. Collectively, our results highlight the ability of PEDV to stimulate the generation of IgA, particularly in elder pigs, and identify TLR9 as a critical mediator of IgA production within the porcine intestinal microenvironment during PEDV infection.
Subject(s)
Coronavirus Infections , Immunoglobulin A , Porcine epidemic diarrhea virus , Toll-Like Receptor 9 , Animals , B-Lymphocytes/immunology , Coculture Techniques , Coronavirus Infections/veterinary , Coronavirus Infections/immunology , Coronavirus Infections/virology , Dendritic Cells/immunology , Immunoglobulin A/immunology , Intestine, Small/immunology , Porcine epidemic diarrhea virus/immunology , Swine , Swine Diseases/immunology , Swine Diseases/virology , Toll-Like Receptor 9/metabolism , Toll-Like Receptor 9/geneticsABSTRACT
Recombinant adeno-associated viruses (AAVs) allow rapid and efficient gene delivery to the nervous system, are widely used in neuroscience research, and are the basis of FDA-approved neuron-targeting gene therapies. Here we find that an innate immune response to the AAV genome reduces dendritic length and complexity and disrupts synaptic transmission in mouse somatosensory cortex. Dendritic loss is apparent 3 weeks after injection of experimentally relevant viral titers, is not restricted to a particular capsid serotype, transgene, promoter, or production facility, and cannot be explained by responses to surgery or transgene expression. AAV-associated dendritic loss is accompanied by a decrease in the frequency and amplitude of miniature excitatory postsynaptic currents and an increase in the proportion of GluA2-lacking, calcium-permeable AMPA receptors. The AAV genome is rich in unmethylated CpG DNA, which is recognized by the innate immunoreceptor Toll-like receptor 9 (TLR9), and acutely blocking TLR9 preserves dendritic complexity and AMPA receptor subunit composition in AAV-injected mice. These results reveal unexpected impacts of an immune response to the AAV genome on neuronal structure and function and identify approaches to improve the safety and efficacy of AAV-mediated gene delivery in the nervous system.
Subject(s)
Dendrites , Dependovirus , Genetic Vectors , Immunity, Innate , Synaptic Transmission , Toll-Like Receptor 9 , Animals , Dependovirus/genetics , Mice , Dendrites/metabolism , Toll-Like Receptor 9/metabolism , Toll-Like Receptor 9/genetics , Genetic Vectors/genetics , Genetic Vectors/administration & dosage , Receptors, AMPA/genetics , Receptors, AMPA/metabolism , Somatosensory Cortex/metabolism , Somatosensory Cortex/immunology , Genome, ViralABSTRACT
BACKGROUND: Toll-like receptors (TLRs) play a critical role in initiating the innate immune response to infection or injury. Recent studies have uncovered their intriguing functions as moonlighting proteins involved in various biological processes, including development, learning, and memory. However, the specific functions of individual TLRs are still largely unknown. AIMS: We investigated the effects of TLR3 and TLR9 receptor deficiency on motor, cognitive, and behavioral functions during development using genetically modified male mice of different ages. METHODS: We evaluated the motor coordination, anxiety-like behavior, spatial learning, and working memory of male mice lacking the TLR3 and TLR9 genes at different ages (two, four, six, and eight months) using the rotarod, open field, water maze, and T-maze tests. RESULTS: We observed that the deletion of either TLR3 or TLR9 resulted in impaired motor performance. Furthermore, young TLR3-deficient mice exhibited reduced anxiety-like behavior and spatial learning deficits; however, their working memory was unaffected. In contrast, young TLR9-knockout mice showed hyperactivity and a tendency toward decreased working memory. CONCLUSIONS: These findings provide valuable insights into the broader roles of the TLR system beyond the innate immune response, revealing its involvement in pathways associated with the central nervous system. Importantly, our results establish a strong association between the endosomal receptors TLR3 and TLR9 and the performance of motor, cognitive, and behavioral tasks that change over time. This study contributes to the growing body of research on the multifaceted functions of TLRs and enhances our understanding of their participation in non-immune-related processes.
Subject(s)
Toll-Like Receptor 3 , Toll-Like Receptor 9 , Animals , Male , Mice , Cognition , Mice, Knockout , Toll-Like Receptor 3/genetics , Toll-Like Receptor 3/metabolism , Toll-Like Receptor 9/genetics , Toll-Like Receptors/genetics , Toll-Like Receptors/metabolismABSTRACT
Herpes simplex virus 1 (HSV-1) or simplexvirus humanalpha 1 is a neurotropic virus that is responsible for orofacial infections in humans. More than 70% of the world's population may have seropositivity for HSV-1, and this virus is a leading cause of sporadic lethal encephalitis in humans. The role of toll-like receptors (TLRs) in defending against HSV-1 infection has been explored, including the consequences of lacking these receptors or other proteins in the TLR pathway. Cell and mouse models have been used to study the importance of these receptors in combating HSV-1, how they relate to the innate immune response, and how they participate in the orchestration of the adaptive immune response. Myeloid differentiation factor 88 (MyD88) is a protein involved in the downstream activation of TLRs and plays a crucial role in this signaling. Mice with functional MyD88 or TLR2 and TLR9 can survive HSV-1 infection. However, they can develop encephalitis and face a 100% mortality rate in a dose-dependent manner when MyD88 or TLR2 plus TLR9 proteins are non-functional. In TLR2/9 knockout mice, an increase in chemokines and decreases in nitric oxide (NO), interferon (IFN) gamma, and interleukin 1 (IL-1) levels in the trigeminal ganglia (TG) have been correlated with mortality.
Subject(s)
Encephalitis , Herpes Simplex , Herpesvirus 1, Human , Humans , Animals , Mice , Herpesvirus 1, Human/metabolism , Toll-Like Receptor 2/metabolism , Toll-Like Receptor 9/genetics , Toll-Like Receptor 9/metabolism , Myeloid Differentiation Factor 88/genetics , Myeloid Differentiation Factor 88/metabolism , Trigeminal Ganglion/metabolism , Toll-Like Receptors/metabolism , Mice, Knockout , Mice, Inbred C57BLABSTRACT
As hippocampal neurons respond to diverse types of information1, a subset assembles into microcircuits representing a memory2. Those neurons typically undergo energy-intensive molecular adaptations, occasionally resulting in transient DNA damage3-5. Here we found discrete clusters of excitatory hippocampal CA1 neurons with persistent double-stranded DNA (dsDNA) breaks, nuclear envelope ruptures and perinuclear release of histone and dsDNA fragments hours after learning. Following these early events, some neurons acquired an inflammatory phenotype involving activation of TLR9 signalling and accumulation of centrosomal DNA damage repair complexes6. Neuron-specific knockdown of Tlr9 impaired memory while blunting contextual fear conditioning-induced changes of gene expression in specific clusters of excitatory CA1 neurons. Notably, TLR9 had an essential role in centrosome function, including DNA damage repair, ciliogenesis and build-up of perineuronal nets. We demonstrate a novel cascade of learning-induced molecular events in discrete neuronal clusters undergoing dsDNA damage and TLR9-mediated repair, resulting in their recruitment to memory circuits. With compromised TLR9 function, this fundamental memory mechanism becomes a gateway to genomic instability and cognitive impairments implicated in accelerated senescence, psychiatric disorders and neurodegenerative disorders. Maintaining the integrity of TLR9 inflammatory signalling thus emerges as a promising preventive strategy for neurocognitive deficits.
Subject(s)
CA1 Region, Hippocampal , DNA Breaks, Double-Stranded , DNA Repair , Inflammation , Memory , Toll-Like Receptor 9 , Animals , Female , Male , Mice , Aging/genetics , Aging/pathology , CA1 Region, Hippocampal/physiology , Centrosome/metabolism , Cognitive Dysfunction/genetics , Conditioning, Classical , Extracellular Matrix/metabolism , Fear , Genomic Instability/genetics , Histones/metabolism , Inflammation/genetics , Inflammation/immunology , Inflammation/metabolism , Inflammation/pathology , Memory/physiology , Mental Disorders/genetics , Neurodegenerative Diseases/genetics , Neuroinflammatory Diseases/genetics , Neurons/metabolism , Neurons/pathology , Nuclear Envelope/pathology , Toll-Like Receptor 9/deficiency , Toll-Like Receptor 9/genetics , Toll-Like Receptor 9/immunology , Toll-Like Receptor 9/metabolismABSTRACT
Epstein-Barr virus (EBV) is a human tumor virus associated with a variety of malignancies, including nasopharyngeal carcinoma, gastric cancers, and B-cell lymphomas. N6-methyladenosine (m6A) modifications modulate a wide range of cellular processes and participate in the regulation of virus-host cell interactions. Here, we discovered that EBV infection downregulates toll-like receptor 9 (TLR9) m6A modification levels and thus inhibits TLR9 expression. TLR9 has multiple m6A modification sites. Knockdown of METTL3, an m6A "writer", decreases TLR9 protein expression by inhibiting its mRNA stability. Mechanistically, Epstein-Barr nuclear antigen 1 increases METTL3 protein degradation via K48-linked ubiquitin-proteasome pathway. Additionally, YTHDF1 was identified as an m6A "reader" of TLR9, enhancing TLR9 expression by promoting mRNA translation in an m6A -dependent manner, which suggests that EBV inhibits TLR9 translation by "hijacking" host m6A modification mechanism. Using the METTL3 inhibitor STM2457 inhibits TLR9-induced B cell proliferation and immunoglobulin secretion, and opposes TLR9-induced immune responses to assist tumor cell immune escape. In clinical lymphoma samples, the expression of METTL3, YTHDF1, and TLR9 was highly correlated with immune cells infiltration. This study reveals a novel mechanism that EBV represses the important innate immunity molecule TLR9 through modulating the host m6A modification system.
Subject(s)
Adenosine , Herpesvirus 4, Human , Methyltransferases , RNA-Binding Proteins , Toll-Like Receptor 9 , Humans , Adenosine/analogs & derivatives , Adenosine/metabolism , B-Lymphocytes/metabolism , B-Lymphocytes/immunology , B-Lymphocytes/virology , Epstein-Barr Virus Infections/immunology , Epstein-Barr Virus Infections/metabolism , Epstein-Barr Virus Infections/virology , Epstein-Barr Virus Infections/genetics , Herpesvirus 4, Human/immunology , Herpesvirus 4, Human/genetics , Herpesvirus 4, Human/metabolism , Immune Evasion , Methyltransferases/metabolism , Methyltransferases/genetics , RNA-Binding Proteins/metabolism , RNA-Binding Proteins/genetics , RNA-Binding Proteins/immunology , Toll-Like Receptor 9/metabolism , Toll-Like Receptor 9/genetics , Cell Line, TumorABSTRACT
The transmembrane pattern recognition receptor, Toll-like receptor (TLR), are best known for their roles in innate immunity via recognition of pathogen and initiation of signaling response. Mammalian TLRs recognize molecular patterns associated with pathogens and initiate innate immune response. We have studied the evolutionary diversity of mammalian TLR genes for differences in immunological response. Reconstruction of ancestral sequences is a key aspect of the molecular evolution of TLR to track changes across the TLR genes. The comprehensive analysis of mammalian TLRs revealed a distinct pattern of evolution of TLR9. Various sequence-based features such as amino acid usage, hydrophobicity, GC content, and evolutionary constraints are found to influence the divergence of TLR9 from other TLRs. Ancestral sequence reconstruction analysis also revealed that the gradual evolution of TLR genes in several ancestral lineages leads to the distinct pattern of TLR9. It demonstrates evolutionary divergence with the progressive accumulation of mutations results in the distinct pattern of TLR9.
Subject(s)
Evolution, Molecular , Phylogeny , Toll-Like Receptor 9 , Toll-Like Receptor 9/genetics , Animals , Humans , Genetic Variation , Amino Acid Sequence , Base CompositionABSTRACT
Sepsis is a life-threatening organ dysfunction due to dysregulated host response to infection, accompanied by a high rate of mortality worldwide. During sepsis progression, toll-like receptors (TLRs) play essential roles in the aberrant inflammatory response that contributes to sepsis-related mortality. Here, we demonstrated a critical role of TLR9 in the progression of sepsis. A septic mouse model was established by cecal ligation and puncture (CLP), then administered with lentivirus encoding si-TLR9/LY294002. TLR9 protein expression and p65 nuclear translocation level/TLR9 protein positive expression/interaction between TLR9 and myeloid differentiation primary response protein 88 (MyD88) in the cecal tissues were examined by Western blot/immunohistochemistry/co-immunoprecipitation assays. Serum levels of pro-inflammatory factors [e.g., interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α)] as well as bacterial contents in the liver/spleen/mesenteric lymph nodes (MLN) were measured by ELISA and bacterial mobility assay. TLR9 expression was augmented in the cecal tissues, TLR9 and MyD88 interaction was enhanced, nuclear p65 protein level was increased, cytoplasmic p65 protein level was decreased, and the nuclear factor kappa B (NF-κB) pathway was activated in CLP-induced septic mice, while TLR9 knockout protected against CLP-induced sepsis via the MyD88/NF-κB pathway inactivation. Briefly, TLR9 inhibition-mediated protection against CLP-induced sepsis was associated with a reduction in pro-inflammatory cytokine release and a promotion of bacterial clearance via a mechanism involving the MyD88/NF-κB pathway inactivation.